The present invention relates to a very remarkable improvement in the in vivo transfer of nucleic acids into the cells of pluricellular eukaryotic organisms or of nucleic acids combined with products which make it possible to increase the yield of such transfers using weak electric fields of between 1 and 600 V/cm, and to the combination of a nucleic acid and the method of transfer according to the invention for their use in gene therapy.
The transfer of genes into a given cell is at the root of gene therapy. However, one of the problems is to succeed in causing a sufficient quantity of nucleic acid to penetrate into cells of the host to be treated; indeed, this nucleic acid, in general a gene of interest, has to be expressed in transfected cells. One of the approaches selected in this regard has been the integration of the nucleic acid into viral vectors, in particular into retroviruses, adenoviruses or adeno-associated viruses. These systems take advantage of the cell penetration mechanisms developed by viruses, as well as their protection against degradation. However, this approach has disadvantages, and in particular a risk of production of infectious viral particles capable of dissemination in the host organism, and, in the case of retroviral vectors, a risk of insertional mutagenesis. Furthermore, the capacity for insertion of a therapeutic or vaccinal gene into a viral genome remains limited.
In any case, the development of viral vectors capable of being used in gene therapy requires the use of complex techniques for defective viruses and for complementation cell lines.
Another approach (Wolf et al. Science 247, 1465-68, 1990; Davis et al. Proc. Natl. Acad. Sci. USA 93, 7213-18, 1996) has therefore consisted in administering into the muscle or into the blood stream a nucleic acid of a plasmid nature, combined or otherwise with compounds intended to promote its transfection, such as proteins, liposomes, charged lipids or cationic polymers such as polyethylenimine, which are good transfection agents in vitro (Behr et al. Proc. Natl. Acad. Sci. USA 86, 6982-6, 1989; Felgner et al. Proc. Natl. Acad. Sci. USA 84, 7413-7, 1987; Boussif et al. Proc. Natl. Acad. Sci. USA 92, 7297-301, 1995).
As regards the muscle, since the initial publication by J. A. Wolff et al. showing the capacity of muscle tissue to incorporate DNA injected in free plasmid form (Wolff et al. Science 247, 1465-1468, 1990), numerous authors have tried to improve this procedure (Manthorpe et al., 1993, Human Gene Ther. 4, 419-431; Wolff et al., 1991, BioTechniques 11, 474-485). A few trends emerge from these tests, such as in particular:
the use of mechanical solutions to force the entry of DNA into cells by adsorbing the DNA onto beads which are then propelled onto the tissues (xe2x80x9cgene gunxe2x80x9d) (Sanders Williams et al., 1991, Proc. Natl. Acad. Sci. USA 88, 2726-2730; Fynan et al., 1993, BioTechniques 11, 474-485). These methods have proved effective in vaccination strategies but they affect only the top layers of the tissues. In the case of the muscle, their use would require a surgical approach in order to allow access to the muscle because the particles do not cross the skin tissues;
the injection of DNA, no longer in free plasmid form but combined with molecules capable of serving as vehicle facilitating the entry of the complexes into cells. Cationic lipids, which are used in numerous other transfection methods, have proved up until now disappointing, because those which have been tested have been found to inhibit transfection (Schwartz et al., 1996, Gene Ther. 3, 405-411). The same applies to cationic peptides and polymers (Manthorpe et al., 1993, Human Gene Ther. 4, 419-431). The only case of a favourable combination appears to be the mixing of poly(vinyl alcohol) or polyvinylpyrrolidone with DNA. The increase resulting from these combinations only represents a factor of less than 10 compared with DNA injected in naked form (Mumper et al., 1996, Pharmaceutical Research 13, 701-709);
the pretreatment of the tissue to be injected with solutions intended to improve the diffusion and/or the stability of DNA (Davis et al., 1993, Hum. Gene Ther. 4, 151-159), or to promote the entry of nucleic acids, for example the induction of cell multiplication or regeneration phenomena. The treatments have involved in particular the use of local anaesthetics or of cardiotoxin, of vasoconstrictors, of endotoxin or of other molecules (Manthorpe et al., 1993, Human Gene Ther. 4, 419-431; Danko et al., 1994, Gene Ther. 1, 114-121; Vitadello et al., 1994, Hum. Gene Ther. 5, 11-18). These pretreatment protocols are difficult to manage, bupivacaine in particular requiring, in order to be effective, being injected at doses very close to lethal doses. The preinjection of hyperosmotic sucrose, intended to improve diffusion, does not increase the transfection level in the muscle (Davis et al., 1993).
Other tissues have been transfected in vivo either using plasmid DNA alone or in combination with synthetic vectors (reviews by Cotten and Wagner (1994), Current Opinion in Biotechnology 4, 705; Gao and Huang (1995), Gene Therapy, 2, 710; Ledley (1995), Human Gene Therapy 6, 1129). The principal tissues studied were the liver, the respiratory epithelium, the wall of the vessels, the central nervous system and tumours. In all these tissues, the levels of expression of the transgenes have proved to be too low to envisage a therapeutic application (for example in the liver, Chao et al. (1996) Human Gene Therapy 7, 901), although some encouraging results have recently been obtained for the transfer of plasmid DNA into the vascular wall (Iires et al. (1996) Human Gene Therapy 7,959 and 989). In the brain, the transfer efficiency is very low, likewise in tumours (Schwartz et al. 1996, Gene Therapy 3, 405; Lu et al. 1994, Cancer Gene Therapy 1, 245; Son et al. Proc. Natl. Acad. Sci. USA 91, 12669).
Electroporation, or use of electric fields to permeabilize cells, is also used in vitro to promote the transfection of DNA into cells in culture. However, it has up until now been accepted that this phenomenon responded to an effect which is dependent on a threshold and that this electropermeabilization could only be observed for electric fields of relatively high intensity, of the order of 800 to 1200 volts/cm for animal cells. This technique has also been proposed in vivo to improve the efficacy of antitumour agents, such as bleomycin, in solid tumours in man (American Patent No. 5, 468,228, L. M. Mir). With pulses of very short duration (100 microseconds), these electrical conditions (800 to 1200 volts/cm) are very well suited to the intracellular transfer of small molecules. These conditions (pulses of 100 microseconds) have been applied with no improvement for the transfer of nucleic acids in vivo into the liver, where fields of less than 1000 volts/cm have proved completely ineffective, and even inhibitory compared with the injection of DNA in the absence of electrical impulses (Patent WO 97/07826 and Heller et al. FEBS Letters, 389, 225-8, 1996).
There are in fact difficulties with applying this technique in vivo because the administration of fields of such an intensity may cause extensive tissue lesions to a greater or lesser extent which do not represent a problem for the treatment of cancer patients but which may have a major disadvantage for the healthy subject or the sick subject when the nucleic acid is administered into tissues other than tumour tissues.
Whereas all the studies cited mention the need for high electric fields, of the order of 1000 volts/cm, to be effective in vivo, in a truly unexpected and remarkable manner, the applicants have now shown that the transfer of nucleic acids into tissues in vivo could be very substantially increased, without undesirable effects, by subjecting the tissue to electrical pulses of low intensity, for example 100 or 200 volts/cm and of a relatively long duration. Furthermore, the applicants have observed that the high variability in the expression of the transgene observed in the prior art for the transfer of DNA was notably reduced by the method according to the invention.
Accordingly, the present invention relates to a method of transferring nucleic acids in vivo, in which the cells of the tissues are brought into contact with the nucleic acid to be transferred, by direct administration into the tissue or by topical or systemic administration, and in which the transfer is brought about by application to the said tissues of one or more electrical pulses of an intensity between 1 and 600 volts/cm.
According to a preferred mode, the method according to the invention applies to tissues whose cells have specific geometries, such as for example cells of large size and/or of elongated shape and/or naturally responding to electrical action potentials and/or having a specific morphology.
Preferably, the intensity of the field is between 200 and 600 volts/cm and the total duration of application is greater than 10 milliseconds. The number of pulses used is, for example, from 1 to 100,000 pulses and the frequency of the pulses is between 0.1 and 1000 Hertz. Preferably, the frequency of the pulses is between 0.2 and 100 Hertz. The pulses may also be delivered in an irregular manner and the function which describes the intensity of the field as a function of time may be variable. By way of example, the electric field delivered may result from the combination of at least one field of an intensity  greater than 400 V/cm and preferably of between 500 and 800 Volts/cm, of short unit duration ( less than 1 msec), followed by one or more pulses of lower intensity, for example  less than 400 Volts/cm, and preferably  less than 200 Volts/cm and of longer unit duration ( greater than 1 msec). The integral of the function describing the variation of the electric field with time is greater than 1 kVxc3x97msec/cm. According to a preferred mode of the invention, this integral is greater than or equal to 5 kVxc3x97msec/cm.
According to a preferred mode of the invention, the field intensity of the pulses is approximately 500 volts/cm (i.e. xc2x110% and preferably xc2x15%).
The electrical pulses are chosen from square wave pulses, electric fields generating exponentially decreasing waves, oscillating unipolar waves of limited duration, oscillating bipolar waves of limited duration, or other wave forms. According to a preferred mode of the invention, the electrical pulses are square wave pulses.
The administration of electrical pulses may be carried out by any method known to persons skilled in the art, for example:
system of external electrodes placed on either side of the tissue to be treated, in particular non-invasive electrodes placed in contact with the skin,
system of electrodes implanted in the tissues,
system of electrodes/injector allowing the simultaneous administration of the nucleic acids and the electric field.
Within the framework of the present invention, the terms transfer of DNA or of nucleic acids by application of one or more electrical pulses, as well as the terms electrotransfer or alternatively electrotransfection should be considered as equivalent and designate the transfer of nucleic acids or of DNA by application or in the presence of an electric field.
The administration being carried out in vivo, it is sometimes necessary to use intermediate products which provide electrical continuity with non-invasive external electrodes. This may be for example an electrolyte in gel form.
The nucleic acids may be administered by any appropriate means, but are preferably injected in vivo directly into the tissues or administered by another route, local or systemic and in particular by means of a catheter, which makes them available at the site of application of the electric field. The nucleic acids may be administered with agents allowing or facilitating transfer, as was mentioned above. In particular, these nucleic acids may be free in solution or combined with synthetic agents, or carried by viral vectors. The synthetic agents may be lipids or polymers known to a person skilled in the art, or alternatively targeting elements allowing attachment to the membrane of the target tissues. Among these elements, there may be mentioned vectors carrying sugars, peptides, antibodies or hormone receptors.
It can be understood, under these conditions of the invention, that the administration of the nucleic acids can be preceded by, simultaneous with or even subsequent to the application of the electric fields.
Accordingly, the subject of the present invention is also a nucleic acid and an electric field of an intensity between 1 and 600 volts/cm, as combination product for their administration simultaneously, separately or spaced out over time, to mammalian cells and in particular human cells, in vivo. Preferably, the intensity of the field is between 200 and 600 volts/cm and, more preferably still, the intensity of the field is approximately 500 volts/cm.
The method according to the present invention can be used in gene therapy, that is to say therapy in which the expression of a transferred gene, but also the modulation or the blocking of a gene, makes it possible to provide the treatment of a particular pathological condition.
Preferably, the cells of the tissues are treated for the purpose of a gene therapy allowing:
either the correction of dysfunctions of the cells themselves (for example for the treatment of diseases linked to genetic deficiencies such as for example cystic fibrosis),
or the safeguard and/or the regeneration of the vascularization or the innervation of the tissues or organs by trophic, neurotrophic and angiogenic factors produced by the transgene,
or the transformation of the tissue into an organ secreting products leading to a therapeutic effect such as the product of the gene itself (for example factors for regulation of thrombosis and of haemostasis, trophic factors, hormones) or such as an active metabolite synthesized in the tissue by virtue of the addition of the therapeutic gene,
or a vaccine or immunostimulant application.
Another subject of the invention is the combination of the electrical pulses of a field with compositions containing nucleic acids formulated for any administration allowing access to the tissue by the topical, cutaneous, oral, vaginal, parenteral, intranasal, intravenous, intra-arterial, intramuscular, subcutaneous, intraocular or transdermal route, and the like. Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection into the desired organ, or for any other administration. They may be in particular isotonic sterile solutions or dry, in particular freeze-dried, compositions which, upon addition, depending on the case, of sterilized water or of physiological saline, allow the preparation of injectable solutions. The nucleic acid doses used for the injection as well as the number of administrations and the volume of injections may be adjusted according to various parameters, and in particular according to the mode of administration used, the relevant pathological condition, the gene to be expressed, or the desired duration of treatment.
The nucleic acids may be of synthetic or biosynthetic origin, or may be extracted from viruses or prokaryotic cells or from eukaryotic cells derived from unicellular organisms (for example yeasts) or from pluricellular organisms. They may be administered in combination with all or part of the components of the organism of origin and/or of the synthesis system.
The nucleic acid may be a deoxyribonucleic acid or a ribonucleic acid. It may be sequences of natural or artificial origin, and in particular genomic DNA, cDNA, mRNA, tRNA and rRNA, hybrid sequences or synthetic or semisynthetic sequences of modified or unmodified oligonucleotides. These nucleic acids may be obtained by any technique known to persons skilled in the art, and in particular by targeting libraries, by chemical synthesis or by mixed methods including chemical or enzymatic modification of sequences obtained by targeting libraries. They may be chemically modified.
In particular, the nucleic acid may be a DNA or a sense or antisense RNA or an RNA having catalytic property such as a ribozyme. xe2x80x9cAntisensexe2x80x9d is understood to mean a nucleic acid having a sequence complementary to a target sequence, for example an mRNA sequence the blocking of whose expression is sought by hybridization with the target sequence. xe2x80x9cSensexe2x80x9d is understood to mean a nucleic acid having a sequence which is homologous or identical to a target sequence, for example a sequence which binds to a protein transcription factor and which is involved in the expression of a given gene. According to a preferred embodiment, the nucleic acid comprises a gene of interest and elements allowing the expression of the said gene of interest. Advantageously, the nucleic acid fragment is in the form of a plasmid.
The deoxyribonucleic acids may be single- or double-stranded, as well as short oligonucleotides or longer sequences. They may carry therapeutic genes, sequences for regulation of transcription or of replication, or regions for binding to other cellular components, and the like. For the purposes of the invention, xe2x80x9ctherapeutic genexe2x80x9d is understood to mean in particular any gene encoding an RNA or a protein product having a therapeutic effect. The protein product encoded may be a protein, a peptide and the like. This protein product may be homologous in relation to the target cell (that is to say a product which is normally expressed in the target cell when the latter exhibits no pathological condition). In this case, the expression of the transgene makes it possible, for example, to overcome an inadequate expression in the cell or the expression of an inactive or weakly active protein due to a modification, or makes it possible to overexpress the said protein. The therapeutic gene may also encode a mutant of a cellular protein having increased stability or a modified activity, and the like. The protein product may also be heterologous in relation to the target cell. In this case, an expressed protein may, for example, supplement or provide an activity which is deficient in the cell (treatment of enzymatic deficiencies), or may make it possible to combat a pathological condition, or to stimulate an immune response for example for the treatment of tumours. It may be a suicide gene (Herpes Thymidine Kinase) for the treatment of cancers or of restenosis.
Among the therapeutic products for the purposes of the present invention, there may be mentioned more particularly the genes encoding
enzymes, such as xcex1-1-antitrypsin, proteinase (metalloproteinases, urokinase, uPA, tPA, . . . streptokinase), proteases cleaving precursors in order to liberate active products (ACE, ICE, . . . ), or their antagonists (TIMP-1, tissue plasminogen activator inhibitor PAI, TFPI
blood derivatives such as the factors involved in coagulation: factors VII, VIII, IX, complement factors, thrombin,
hormones, or enzymes involved in the pathway for the synthesis of hormones, or factors involved in controlling the synthesis or the excretion or the secretion of hormones, such as insulin, factors close to insulin (IGF), or growth hormone, ACTH, enzymes for the synthesis of sex hormones,
lymphokines and cytokines: interleukins, chemokines (CXC and CC), interferons, TNF, TGF, chemotactic factors or activators such as MIF, MAF, PAF, MCP-1, eotaxin, LIF, and the like (French Patent No. 92 03120),
growth factors, for example IGF, EGF, FGF, KGF, NGF, PDGF, PIGF, HGF, proliferin
angiogenic factors such as VEGF of FGF, angiopoietin 1 or 2, endothelin
enzymes for synthesizing neurotransmitters,
trophic factors, in particular neurotrophic factors for the treatment of neurodegenerative diseases, traumas which have damaged the nervous system, or retinal degeneration, such as members of the family of neurotrophins such as NGF, BDNF, NT3, NT4/5, NT6, their derivatives and related genesxe2x80x94members of the CNTF families such as CNTF, axokine, LIF and derivatives thereofxe2x80x94IL6 and its derivativesxe2x80x94cardiotrophin and its derivativesxe2x80x94GDNF and its derivativesxe2x80x94members of the family of IGFs, such as IGF-1, IFGF-2 and derivatives thereofxe2x80x94members of the FGF family, such as FGF 1, 2, 3, 4, 5, 6, 7, 8, 9 and derivatives thereof, TGFxcex2
bone growth factors,
haematopoietic factors, such as erythropoietin, GM-CSF, M-CSF, LIF, and the like,
cellular architectural proteins such as dystrophin or minidystrophin (French Patent No. 91 11947),, suicide genes (thymidine kinase, cytosine deaminase, cytochrome P450-containing enzymes), genes for haemoglobin or other protein carriers,
genes corresponding to the proteins involved in the metabolism of lipids, of the apolipoprotein type chosen from apolipoproteins A-I, A-II, A-IV, B, C-I, C-II, C-III, D, E, F, G, H, J and apo(a), metabolic enzymes such as, for example, lipases, lipoprotein lipase, hepatic lipase, lecithin-cholesterol acyltransferase, 7-alpha-cholesterol hydroxylase, phosphatidyl acid phosphatase, or alternatively lipid transfer proteins such as the cholesterol ester transfer protein and the phospholipid transfer protein, an HDL-binding protein or alternatively a receptor chosen, for example, from the LDL receptors, the remnant chylomicron receptors and the scavenger receptors, and the like. It is furthermore possible to add leptin for the treatment of obesity.
blood pressure regulating factors, such as the enzymes involved in the metabolism of NO, angiotensin, bradykinin, vasopressin, ACE, renin, the enzymes encoding the mechanisms for the synthesis or for the relief of prostaglandins, thromboxan, or adenosine, adenosine receptors, kallikreins and kallistatins, ANP, ANF, diuretic or antidiuretic factors, factors involved in the synthesis, the metabolism or the release of mediators such as histamine, serotonin, catecholamines, neuropeptides,
anti-angiogenic factors such as the ligand for Tie-1 and for Tie-2, angiostatin, ATF factor, derivatives of plasminogen, endothelin, thrombospondins 1 and 2, PF-4, xcex1- or xcex2-interferon, interleukin-12, TNFxcex1, urokinase receptor, flt1, KDR, PAI1, PAI2, TIMP1, prolactin fragment,
factors protecting against apoptosis, such as the AKT family,
proteins capable of inducing cell death, either active by themselves such as the caspases or of the xe2x80x9cpro-drugxe2x80x9d type requiring activation by other factors, or proteins activating pro-drugs into an agent causing cell death, such as the herpesvirus thymidine kinase, deaminases, which make it possible in particular to envisage anticancer therapies,
proteins involved in intercellular contacts and adhesion: VCAM, PECAM, ELAM, ICAM, integrins, cathenins,
proteins of the extracellular matrix,
proteins involved in the migration of cells
proteins of the signal transduction type, of the type including FAK, MEKK, p38 kinase, tyrosines, kinases, serine-threonine kinases,
proteins involved in the regulation of the cell cycle (p21, p16, cyclines, . . . ) as well as the dominant negative mutant or derived proteins blocking the cell cycle and capable, where appropriate, of inducing apoptosis.
transcription factors: jun, fos, AP1, p53, . . . and the proteins of the p53 signalling cascade.
cell structure proteins, such as the intermediate filaments (vimentin, desmin, keratins), dystrophin, the proteins involved in muscle contractility and in controlling muscle contractibility, in particular the proteins involved in calcium metabolism and the flow of calcium in the cells (SERCA, . . . ).
In the cases of proteins which function through ligand and receptor systems, it is possible to envisage using the ligand or the receptor (e.g. FGF-R, VEGR-R, . . . ). It is also possible to mention genes encoding fragments or mutants of ligand or receptor proteins, in particular of the abovementioned proteins, either having an activity greater than the whole protein, or an antagonist activity, or even an activity of the xe2x80x9cdominant negativexe2x80x9d type relating to the initial protein (for example fragments of receptors inhibiting the availability of circulating proteins, associated or otherwise with sequences inducing secretion of these fragments in relation to anchorage in the cell membrane, or other systems for modifying the intracellular traffic of these ligand-receptor systems so as to divert the availability of one of the elements) or even possessing an inherent activity distinct from that of the total protein (e.g. ATF).
Among the other proteins or peptides which may be secreted by the tissue, it is important to underline antibodies, the variable fragments of single-chain antibody (ScFv) or any other antibody fragment possessing recognition capacities for its use in immunotherapy, for example for the treatment of infectious diseases, of tumours, of autoimmune diseases such as multiple sclerosis (antiidiotype antibodies) as well as the ScFv""s which becomes attached to the pro-inflammatory cytokines such as, for example, IL1 and TNFxcex1 for the treatment of rheumatoid arthritis. Other proteins of interest are, in a nonlimiting manner, soluble receptors such as, for example, the soluble CD4 receptor or the soluble receptor for TNF for anti-HIV therapy, the TNFxcex1 receptor or the IL1 soluble receptor for the treatment of rheumatoid arthritis, the soluble receptor for acetylcholine for the treatment of myasthenia; substrate peptides or enzyme inhibitors, or peptides which are agonists or antagonists of receptors or of adhesion proteins such as, for example, for the treatment of asthma, thrombosis, restenosis, metastasis or inflammation; artificial, chimeric or truncated proteins. Among the hormones of essential interest, there may be mentioned insulin in the case of diabetes, growth hormone and calcitonin. It is also possible to mention proteins capable of inducing antitumour immunity or of stimulating the immune response (IL2, GM-CSF, IL12, and the like). Finally, it is possible to mention the cytokines which reduce the TH1 response such as IL10, IL4 and Il13.
The numerous examples which precede and those which follow illustrate the potential scope of the field of application of the present invention.
The therapeutic nucleic acid may also be an antisense sequence or gene whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences may, for example, be transcribed in the target cell into RNA complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in European Patent No. 140 308. The therapeutic genes also comprise the sequences encoding ribozymes, which are capable of selectively destroying target RNAs (European Patent No. 321 201).
As indicated above, the nucleic acid may also comprise one or more genes encoding an antigenic peptide capable of generating an immune response in humans or in animals. In this particular embodiment, the invention therefore allows either the production of vaccines, or the carrying out of immunotherapeutic treatments applied to humans or to animals, in particular against microorganisms, viruses or cancers. It may be in particular antigenic peptides specific for the Epstein-Barr virus, the HIV virus, the hepatitis B virus (European Patent No. 185 573), the pseudo-rabies virus, the xe2x80x9csyncytia forming virusxe2x80x9d, other viruses or antigens specific for tumours such as the MAGE proteins (European Patent No. 259 212), such as MAGE 1, MAGE 2 proteins or antigens which can stimulate an anti-tumour response such as bacterial heat shock proteins.
Preferably, the nucleic acid also comprises sequences allowing and/or promoting the expression, in the tissue, of the therapeutic gene and/or of the gene encoding the antigenic peptide. They may be sequences which are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the transfected cell. They may also be sequences of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences derived from the genome of the cell which it is desired to transfect. Among the eukaryotic promoters, there may be mentioned any promoter or derived sequence stimulating or repressing the transcription of a gene in a specific manner or otherwise, strongly or weakly. They may be in particular ubiquitous promoters (HPRT, vimentin, xcex1-actin, tubulin, and the like), promoters of therapeutic genes (of the type including MDR, CFTR, and the like), tissue-specific promoters (of the type including promoters of genes for desmin, myosins, creatine kinase, phosphoglycerate kinase) or alternatively promoters responding to a stimulus such as promoters responding to the natural hormones (receptor for steroid hormones, receptor for retinoic acid, and the like) or a promoter regulated by antibiotics (tetracyclin, rapamycin, and the like), promoters responding to a dietary regimen such as the promoters responding to fibrates, or other promoters responding to other molecules of natural or synthetic origin. Likewise, they may be promoter sequences derived from the genome of a virus. In this regard, there may be mentioned, for example, the promoters of the EIA genes of the adenovirus, MLP genes, or promoters derived from genomes of the viruses CMV, RSV, SV40, and the like. The promoters may also be inducible or repressible. In addition, these expression sequences may be modified by the addition of activating or regulatory sequences, allowing a conditional or transient expression, a tissue-specific or predominant expression, and the like.
Moreover, the nucleic acid may also comprise, in particular upstream of the therapeutic gene, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence. The nucleic acid may also comprise a signal sequence directing the synthesized therapeutic product towards a particular compartment of the cell, such as, for example, peroxisomes, lysosomes and mitochondria for the treatment, for example, of mitochondrial genetic diseases.
Other genes which are of interest have been described in particular by McKusick, V. A. Mendelian (Inheritance in man, catalogs of autosomal dominant, autosomal recessive, and X-linked phenotypes. Eighth edition, John Hopkins University Press (1988)), and in Stanbury, J. B. et al. (The metabolic basis of inherited disease, Fifth edition, McGraw-Hill (1983)). The genes of interest cover the proteins involved in the metabolism of amino acids, lipids and other constituents of the cell.
There may thus be mentioned, with no limitation being implied, the genes associated with diseases of carbohydrate metabolism such as for example fructose-1-phosphate aldolase, fructose-1,6-diphosphatase, glucose-6-phosphatase, lysosomal xcex1-1,4-glucosidase, amylo-1,6-glucosidase, amylo-(1,4:1,6)-transglucosidase, muscle phosphorylase, muscle phosphofructokinase, phosphorylase-xcex2-kinase, galactose-1-phosphate uridyl transferase, all the enzymes of the complex pyruvate dehydrogenase, pyruvate carboxylase, 2-oxoglutarate glyoxylase carboxylase, D-glycerate dehydrogenase.
There may also be mentioned:
the genes associated with diseases of amino acid metabolism such as for example phenylalanine hydroxylase, dihydrobiopterin synthetase, tyrosine aminotransferase, tyrosinase, histidinase, fumarylacetoacetase, glutathione synthetase, xcex3-glutamylcysteine synthetase, ornithine-xcex4-aminotransferase, carbamoylphosphate synthetase, ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase, arginase, L-lysine dehydrogenase, L-lysine ketoglutarate reductase, valine transaminase, leucine isoleucine transaminase, decarboxylase for the branched-chain 2-keto acids, isovaleryl-CoA dehydrogenase, acyl-CoA dehydrogenase, 3-hydroxy-3-methylglutaryl-CoA lyase, acetoacetyl-CoA 3-ketothiolase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, ATP:cobalamine adenosyltransferase, dihydrofolate reductase, methylenetetrahydrofolate reductase, cystathionine xcex2-synthetase, the sarcosine dehydrogenase complex, proteins belonging to the system for cleaving glycine, xcex2-alanine transaminase, serum carnosinase, cerebral homocarnosinase;
the genes associated with diseases of fat and fatty acid metabolism, such as for example lipoprotein lipase, apolipoprotein C-II, apolipoprotein E, other apolipoproteins, lecithin-cholesterol acyltransferase, LDL receptor, liver sterol hydroxylase, xe2x80x9cphytanic acidxe2x80x9d xcex1-hydroxylase;
the genes associated with lysosomal deficiencies, such as for example lysosomal xcex1-L-iduronidase, lysosomal iduronate sulphatase, lysosomal heparan N-sulphatase, lysosomal N-acetyl-xcex1-D-glucosaminidase, lysosomal acetyl-CoA:xcex1-glucosamine N-acetyltransferase, lysosomal N-acetyl-xcex1-D-glucosamine 6-sulphatase, lysosomal galactosamine 6-sulphate sulphatase, lysosomal xcex2-galactosidase, lysosomal arylsulphatase B, lysosomal xcex2-glucuronidase, N-acetylglucosaminyl-phosphotransferase, lysosomal xcex1-D-mannosidase, lysosomal xcex1-neuraminidase, lysosomal aspartylglycosaminidase, lysosomal xcex1-L-fucosidase, lysosomal acid lipase, lysosomal acid ceramidase, lysosomal sphingomyelinase, lysosomal glucocerebrosidase and lysosomal galactocerebrosidase, lysosomal galactosylceramidase, lysosomal arylsulphatase A, xcex1-galactosidase A, lysosomal acid xcex2-galactosidase, xcex1 chain of lysosomal hexoaminidase A.
There may also be mentioned, in a nonrestrictive manner, the genes associated with diseases of steroid and lipid metabolism, the genes associated with diseases of purine and pyrimidine metabolism, the genes associated with diseases of porphyrin and haem metabolism, the genes associated with diseases of connective tissue, and bone metabolism as well as the genes associated with blood diseases and diseases of the haematopoietic organs, muscle diseases (myopathy), diseases of the nervous system (neurodegenerative diseases) or diseases of the circulatory apparatus (treatment of ischaemias and of stenosis for example) and genes involved in mitochondrial genetic diseases.
In the method according to the invention, the nucleic acid may be combined with any type of vectors or any combination of these vectors which make it possible to improve the transfer of genes, for example, in a nonlimiting manner, with vectors such as viruses, synthetic or biosynthetic agents (for example lipid, polypeptide, glycosidic or polymeric agents), or beads which are propelled or otherwise. The nucleic acids may also be injected into a tissue which has been subjected to a treatment intended to improve the transfer of genes, for example a treatment of a pharmacological nature by local or systemic application, or an enzymatic, permeabilizing (use of surfactants), surgical, mechanical, thermal or physical treatment.
The advantage of the use of electrotransfer in gene therapy lies in the safety provided by the local treatment linked to the use of local and targeted electric fields.
By virtue of the safety linked to the use of weak fields, the present invention could be applied in the region of the cardiac muscle for the treatment of cardiopathies, for example using a suitable defibrillator. It could also be applied to the treatment of restenosis by the expression of genes inhibiting the proliferation of the smooth muscle cells such as the GAX protein.
The combination of fields which are not very intense and which are administered over long periods, applied to the tissues in vivo, improves the transfection of nucleic acids without causing notable damage to the tissues. These results improve the yield of DNA transfers within the context of gene therapy using nucleic acids.
Consequently, the method according to the invention makes it possible, for the first time, to envisage producing, by gene therapy, an agent at physiological and/or therapeutic doses, either in the tissues, or secreted in their vicinity or into the blood stream or the lymph circulation. Furthermore, the method according to the invention allows, for the first time, fine modulation and control of the effective quantity of transgene expressed by the possibility of modulating the volume of tissue to be transfected, for example with multiple sites of administration, or the possibility of modulating the number, the shape, the surface and the arrangement of the electrodes. An additional element of control comes from the possibility of modulating the efficiency of transfection by varying the field intensity, the number, the duration and the frequency of the pulses, and obviously according to the state of the art, the quantity and the volume of nucleic acids to be administered. It is thus possible to obtain an appropriate transfection level at the desired production or secretion level. The method finally allows increased safety compared with the chemical or viral methods for transferring genes in vivo, for which the affecting of organs other than the target organ cannot be completely excluded and controlled. Indeed, the method according to the invention allows control of the localization of the transfected tissues (strictly linked to the volume of tissue subjected to the local electrical pulses) and therefore provides the possibility of a return to the initial situation by complete or partial removal of the tissue when this is made possible by the non-vital character of this tissue and by its regeneration capacities as in the case of the liver or the muscle. This great flexibility of use makes it possible to optimize the method according to the animal species (human and veterinary applications), the age of the subject, his physiological and/or pathological condition.
The method according to the invention makes it possible, in addition, for the first time, to transfect nucleic acids of large size unlike the viral methods which are limited by the size of the capsid. This possibility is essential for the transfer of genes of a very large size such as that for dystrophin or genes with introns and/or regulatory elements of large size, which is necessary for example for a physiologically regulated production of hormones. This possibility is essential for the transfer of episomes or of yeast artificial chromosomes or of minichromosomes.
The following examples are intended to illustrate the invention in a nonlimiting manner.